The overall goal of this proposal is to investigate the molecular target(s) underlying FGF2-mediated cardioprotection. Findings from the original proposal indicate that nitric oxide (NO), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) signaling are necessary for FGF2-induced cardioprotection. Yet, the downstream targets by which these signaling pathways mediate FGF2- induced cardioprotection against myocardial dysfunction and infarction remain to be elucidated. Therefore, we will undertake a pharmacological-, electrophysiological-, subproteomic- and integrative physiological-based multidisciplinary approach to identify known or novel substrates of the signaling pathways associated with FGF2-induced cardioprotection. The proposal will evaluate the involvement of known downstream substrates of kinases that have been affiliated with FGF2 activity or with cardioprotection, and identify novel targets of FGF2-induced cardioprotection by subproteomic analysis. The research plan will integrate basic information at the protein and cellular level with information at the whole organ/animal level. These studies will also enable us to directly relate changes in myocardial infarction and post-ischemic recovery of ventricular function to the biological activity of FGF2 and to specific downstream targets of its protein kinase pathways. To ascertain the involvement of ATP-sensitive potassium (KATP) channels in FGF2-induced cardioprotection, we will employ pharmacologic, electrophysiological, and molecular methods. Similar techniques will be used to determine the importance of FGF2 in regulating calcium homeostasis at the level of sarco(endo)plasmic reticulum (SR) proteins and contractile apparatus, ultimately influencing post- ischemic cardiac function. With preliminary data implicating the mitochondria, SR, and contractile apparatus as targets of FGF2-induced cardioprotection, candidate and novel substrates of these organelles will be interrogated via phosphoproteomic (immunoblotting and 2-D gel/MS) techniques. Results of this proposal will provide new insights into FGF2-induced cardioprotection and may eventually lead to the pharmacologic or genetic development of FGF2 as a therapy against ischemic heart disease. PUBLIC HEALTH RELEVANCE: Cardiovascular disease is the primary cause of death in the United States. Understanding the molecular and cellular basis of cardioprotection is a necessary step towards developing FGF2 as a therapeutic strategy to limit the extent of acute myocardial infarction and protect the heart in the clinical setting.